Recently, arteriosclerosis and coronary atherosclerotic disease (CAD) have been gradually increasing in Korea due to a shift to Western dietary habits, and are the leading cause of increased mortality. Serum lipids causing these diseases include cholesterol, triglycerides (TG), free fatty acids and phospholipids. They form lipoproteins with apolipoproteins and Ere transported through the bloodstream. Among them, low density lipoproteins (LDL) function to transport mainly TG and cholesterol, and changes in LDL-cholesterol levels are indications of the prognosis of the diseases.
LDL-cholesterol, which is a major factor of lipid metabolism-associated diseases of adult people, binds to LDL receptors on the plasma membrane of cells in each tissue and is stored and used in the tissue. Alternatively, LDL-cholesterol is taken up by scavenger cells and hydrolyzed, and free cholesterol is transferred to HDL along with apo E lipoprotein to be recycled in the liver, or is converted to bile salt to be discharged. During this process, the apolipoprotein performs very important functions to maintain structural homeostasis of lipoproteins, serves as a cofactor of the enzyme lipoprotein lipase, and plays a critical role in binding to a specific receptor on the plasma membrane.
Apolipoprotein B-100 (Apo B-100) is a major protein component of LDL, and is also present in IDL and VLDL. Thus, when antibodies in the blood are induced to recognize apo B-100, LDL clearance by phagocytes will easily occur. In this regard, some recent studies have been focused on the employment of vaccines to decrease plasma LDL-cholesterol levels and reduce the incidence of arteriosclerosis. Antibodies induced by such anti-cholesterol vaccine therapy are IgM types which are considered to bind to VLDL, IDL and LDL, and such a strategy suggests the possibility of developing vaccines for preventing and treating hypercholesterolemia and atherosclerosis (Bailey, et al., Cholesterol vaccines. Science 264, 1067-1068, 1994; Palinski W et al., Proc Natl Acad Sci U.S.A. 92, 821-5, 1995; Wu R, de Faire U et al., Hypertension. 33, 53-9, 1999). Also, apolipoprotein B-100 is a huge protein molecule, which consists of 4560 amino acid residues, contains signal peptide of 24 amino acid residues and has a molecular weight of more than 500 kDa (Elovson J et al., Biochemistry, 24:1569-1578, 1985). Since apolipoprotein B-100 is secreted mainly by the liver and is an amphipathic molecule, it can interact with the lipid components of plasma lipoproteins and an aqueous environment (Segrest J. P et al., Adv. Protein Chem., 45:303-369, 1994). Apolipoprotein B-100 stabilizes the size and structure of LDL particles and plays a critical role in controlling the homeostasis of plasma LDL-cholesterol through binding to its receptor (Brown M S et al., Science, 232:34-47, 1986).
Korean Pat. Laid-open Publication No. 2002-0018971, which was filed by the present inventors, describes a mimetic peptide of an epitope of apo B-100 having an anti-obesity effect. However, this publication only discloses that the mimetic peptide of the B cell epitope has an anti-obesity effect.
Prior to the present invention, there is no report of enhancing the immunogenicity of an apolipoprotein by fusing a B cell epitope of the apolipoprotein and a T cell epitope, except for an attempt to enhance immune responses by employing a protein carrier or adjuvant.
As described in U.S. Pat. No. 5,843,446, when luteinizing hormone releasing hormone (LHRH) is conjugated with a different protein to enhance the immunogenicity of LHRH, the majority of immune responses are directed to the carrier protein rather than to LHRH, leading to carrier-induced immune suppression. Thus, persistent effort is required for selecting additional materials and determining linkage patterns and linkage sites capable of enhancing the immunogenicity of B cell epitopes.
Many attempts to fuse a hapten with a carrier protein were made to enhance the immunogenicity of the hapten, but failed to obtain uniform enhancing effects. In particular, the linear linkage of a B cell epitope and a T cell epitope, like the present invention, resulted in loss of immunogenicity according to the orientation of the epitopes, the type of each epitope, and the like (Francis, M. J. et al., Nature 330:168-170, 1987), and the presence of a linker brought about reduced antigenicity (Partidos, C. et al., Mol. Immunol. 29:651-658, 1992). That is, there is no consistent rule applicable to design peptide vaccines, and the efficacy of designed vaccines is also not predictable. For the same reasons, when a highly hydrophobic PB14 peptide, which is an apo-B mimetic peptide, is fused with a T cell epitope, an antigenic region can be internalized into the fusion protein, leading to a decrease in its ability to induce antibody responses.
Based on this background, the present inventors made various attempts to enhance the immunogenicity of PB14, which is a mimetic peptide of a B cell epitope of apolipoprotein B-100 having an anti-obesity effect. As a result, a hybrid polypeptide, in which an N-terminus of a helper T cell epitope is fused to a C-terminus of the mimetic peptide, displayed an excellent immunoenhancing effect, indicating that it is effective for preventing or treating obesity. It was an unexpected result since hybrid polypeptides displays excellent anti-obesity activity without inducing immune responses that neutralize beneficial activities or effects of the B cell epitope of apolipoprotein B-100 or without causing harmful side effects.